Photos courtesy of Meg Perdue, U.S. Fish and Wildlife Service. Studies continue on frogs with limb deformities found on the Kenai National Wildlife Refuge. Observed abnormalities include ectromelia — part of limb missing; amelia — an entire limb missing; and micromelia — a shrunken limb.

By Jenny Neyman

Redoubt Reporter

What’s worse than being a frog — a major link in the food chain and a creature that’s acutely susceptible to deformations from exposure to contaminants or other changes in their environment?

Try being a biologist attempting to tease out definitive conclusions from studying them.

“It’s really hard to nail these things down. That’s what makes science challenging,” said Meg Perdue, a biologist specializing in environmental contaminants for the U.S. Fish and Wildlife Service.

She’s one of the researchers involved in continuing studies of amphibians on national refuges in Alaska, including the Kenai National Wildlife Refuge. The latest iteration of that monitoring project is a study focused specifically on the Kenai in an attempt to discern why, out of the five refuges monitored in the state, the Kenai is showing a higher incidence of frog deformations than any other.

“Out of the monitoring work, when we saw higher rates in Kenai, we focused a study trying to come up with correlations with the malformations,” Perdue said.

This program of amphibian monitoring is a nationwide effort dating back to the mid-1990s when a high number of amphibian deformations — specifically, frogs with extra limbs — was noticed on a refuge in Minnesota.

Frogs are considered a bellwether species, a sort of canary-in-the-coal-mine indicator of changes in an environment, because amphibians are very susceptible to contaminants and other changes in their ecology.

“They tend to bridge multiple environments and their developmental process is out in the environment, unlike mammals and birds that are more protected. Bird eggs at least have a hard shell, and mammals, their embryos are internalized, but frog eggs are just out there in the media and therefore have lot of exposure because of how permeable amphibians are in terms of that interface with the environment,” Perdue said.

So when students on a field trip in Minnesota noticed a preponderance of frogs with multiple limbs, it garnered nationwide focus.

“There was a lot of concern and it got some media attention. That then caused Congress to initiate a program for monitoring so that there could be work done to see if this was an issue in other places around the country,” she said.

In Alaska, the monitoring effort got started in the early 2000s on five national wildlife refuges in the state — Yukon Delta, Innoko, Tetlin, Arctic and Kenai. Much of mainland Alaska only has one resident amphibian species — the wood frog. Monitoring on most of the refuges from about 2000 to 2006 showed 0 percent incidence of abnormalities among frogs. Tetlin showed 3 to 4 percent and Kenai showed 7.8 to 7.9 percent abnormalities.

But unlike the multiple-limb deformations seen in Minnesota, Kenai’s frogs were missing limbs or having asymmetric, underdeveloped limbs. And the cause isn’t the same. In Minnesota, researchers determined that pesticide use and other agricultural practices were the likely causal mechanism of the abnormalities, creating an environment allowing greater algal blooms in which a certain type of parasite can thrive. That parasite, during a period of its life cycle, can burrow into the limb buds of tadpoles and cause them to mutate.

After observing the higher incidence of missing or malformed limbs among Kenai frogs, biologists started trying to determine what might be causing the deformations. To do so, researchers attempted to correlate the incidence of deformities with what else out of the ordinary was going on in the environment. Testing revealed there was not a high parasite load on the Kenai, like was found in Minnesota.

Two environmental factors were noticed in concurrence with the frog deformations — a higher abundance of predators — specifically, dragonfly larvae; and higher levels of inorganic contaminants — specifically, metals such as copper.

So what does that mean? Is copper and associated metals poisonous to frogs? Or is it some kind of superfood to dragonflies? And if there is a link, where is the copper coming from? A quirk of nature, or are humans somehow to blame?

“We wanted to pursue a further study to then see if there was a causal mechanism that sort of involved the predators and inorganics in some sort of mechanism that might lead to the malformations we see there,” Perdue said.

That’s where the biological detective work begins.

v v v

A three-year study was designed and begun in 2010, with fieldwork continuing in 2011 and 2012. The general working hypothesis is that copper affects amphibians in a similarly negative manner as it does salmon. Salmon rely on a highly developed olfactory sense, which allows them to essentially “smell” their way back to their natal stream to spawn, as well as to avoid predators. Studies have shown that even trace amounts of copper introduced in a salmon fry’s habitat can inhibit the fry’s directional sense and ability to sense predators.

Toxicity to frogs has been studied, but primarily only to the extreme. If frogs were dead, it would be fairly straightforward to test for what killed them and stamp a “lethal” label on the culprit. But copper doesn’t outright kill frogs. It’s a question of discovering what sublethal effects it may have.

“How much it alters behavior or differential changes to survivability for an amphibian versus a predator like dragonflies. It might change how they interact,” Perdue said. “It gets very complicated and there are a lot of different variables and so many different interactions in the environment. There’s a huge suite of other factors that could mediate.”

Researchers set up experiments to test the effect of copper on tadpoles. The results were not as dramatic as with salmon fry, but that’s not unexpected, since salmon have a higher sense of olfaction than frogs do.

Perdue said they did discover that the introduction of copper changed tadpole behavior. With salmon fry, copper can cause them to lose the ability to sense predators, and to get directionally lost. The frog tadpoles did still seem to detect the introduction of a predator cue, but didn’t react to it as they ordinarily would — by quickly retreating lower in the water column. Instead, the tadpoles seemed sluggish and didn’t change position a whole lot.

“It appears that what would be nontoxic in a lethal sense seems to change behavior of these tadpoles,” Perdue said. “It changed their behavior but not because they couldn’t detect the predator cue. They could detect that, but with the copper present they weren’t seemingly acting the same way. They were higher in the column or lethargic.”

That reaction could make tadpoles easier prey, which could explain the abundance of dragonfly larvae found in conjunction with the deformed frogs. Perhaps not all the frogs are killed as a result of becoming lazy in predator avoidance. Some may be left mangled with missing or nipped-off limbs.

Or maybe not. That causal link still needs more testing to be confirmed.

“If the tadpoles are acting differently than they normally would be they may be more susceptible to predation attempts because they’re higher up in the water column and more lethargic, and that’s why we’re seeing these abnormalities,” Perdue said. “But that gets complex too because some predators are cued by movement, so lack of movement for some wouldn’t necessarily account for the number of times abnormalities were discovered.”

If copper is found to be the link, there’s still the question of where the copper is coming from. In Washington State, excess copper in the environment that has been found to be harmful to salmon has been attributed to urbanization. Specifically, copper used in automotive brakes. When a driver hits the brakes, friction releases copper shavings onto the road that are eventually washed into the surrounding watershed. In response, Washington State passed a law to phase out the use of copper in brake pads.

Urbanization and the increased traffic it brings could be at least part of the reason for increased levels of copper found in conjunction with frog deformations in Alaska. Only two of the five refuges monitored in the state showed frog abnormalities, and they were the only two that are road accessible. Tetlin, along the Alaska-Canada border, is accessible by the Alaska Highway. And Kenai has even more urban development and roads.

But metals occur naturally in the environment, as well, so it can be challenging to tell if elevated levels of copper are from some kind of human-caused development or simply geology. The Kenai refuge, for instance, also has an abundance of naturally occurring arsenic in the watershed.

“Obviously the amount of development here is a lot less than you have in the Seattle area, so is it sufficient at this point to have an effect or not?” Perdue said.

In the 2000-2006 monitoring, there was no obvious link between roads and frog deformations on the Kenai, Perdue said, but that also wasn’t a relation being specifically tested for. The limb abnormalities were found at sites spread across the refuge — some near roads, others not. In the current study, researchers will test sites strategically to try to gauge the effect of urban development and roads.

In 2010 a pilot study was begun. Testing was conducted this year and will continue next year. Entering and analyzing the data will take time beyond that to see what firm conclusions can be drawn, or if further study is necessary.

The frogs certainly aren’t talking, at least not in language biologists can easily decipher.

“At this point I’m just starting to enter and look at the data from last year. We don’t have anything definitive,” Perdue said. “There are just all of these incredibly complex interactions that are going on. It leaves me scratching my head, I can assure you.”

A careful reading of this article indicates that in Minnesota
“researchers determined that pesticide use and other agricultural practices were the likely causal mechanism of the abnormalities, creating an environment allowing greater algal blooms in which a certain type of parasite can thrive. That parasite, during a period of its life cycle, can burrow into the limb buds of tadpoles and cause them to mutate.” The parasite, in this case, was a trematode. Trematodes occur naturally, however, heavy use of fertilizers, phosphorous and nitrogen led to an increase in their abundance and the higher incidence of observed deformities. See: http://news.minnesota.publicradio.org/features/2005/10/21_bensonl_research/ Since the discovery of deformed frogs in MN similar findings have occurred across the US although different types of deformities have been discovered and different causal factors have been involved. In Kenai’s case, it will likely take scientists awhile to tease out what is causing abnormalities because the pursuit of science, or truth, is time consuming and difficult Best of luck to the researchers and keep searching for the cause!